Detection of specific sequences at candidate loci in target nucleic acid samples is highly important for several reasons relating to diagnosis of inherited disorders and of infectious diseases. Detection of multiple different mutations is necessary for screening for the presence of specific gene tic disorders. Detection of specific sequences in amplified DNA samples significantly enhances current DNA diagnostic methods for the detection of organisms of interest and especially of infectious organisms. Current techniques for the detection of sequences at candidate loci are either cumbersome, lacking in specificity, difficult to optimise and use or poorly adaptable to high sample throughput.
There are several methods known for the detection of a particular nucleic acid sequence at a candidate locus in a target nucleic acid sample. Details of these methods are as follows.
1) Restriction enzyme analysis
Detection of a particular DNA sequence by restriction enzyme analysis. Restriction enzymes cleave DNA at specific sequences. For example, the enzyme EcoRI cleaves double stranded DNA at the sequence GAATTC. Thus, by checking a candidate locus in a particular DNA sample for cleavage with EcoRI, one may deter mine whether the sequence GAATTC is present or absent at the candidate locus. The appearance or disappearance of a restriction site from a candidate locus indicates that one or more of the bases in the sequence at the candidate locus has been altered. Thus, the creation or loss of a restriction site at a candidate locus can involve the alteration of any base or bases of the respective restriction site. Therefore, the appearance or disappearance of a restriction site at a candidate locus may not inform the investigator of the exact sequence change. However, there are a number of problems with the use of restriction enzymes for the detection of specific sequences at candidate loci. These include:
a) the presence or absence of a specific sequence at a candidate locus regularly does not occur at a restriction site; PA1 b) since different restriction enzymes have different recognition sequences, different restriction enzymes are regularly required for the detection of the presence or absence of a particular sequence at different candidate loci; and PA1 c) because different enzymes can be required for the detection of the presence or absence of a particular sequence at different candidate loci, a high throughput is difficult to achieve with this approach and automation is difficult. PA1 a) it is a cumbersome and difficult method for routine detection of the presence or absence of a particular sequence at different candidate loci; PA1 b) full size sequencing gels are required for determination of the presence or absence of a particular sequence at a given candidate locus; PA1 c) the DNA sample for analysis needs to be of high quality in order to obtain good quality DNA sequences; PA1 d) DNA sequencing of directly amplified DNA samples is regularly problematic; PA1 e) sequencing gels can often be difficult to read; PA1 f) high throughput with a high success rate is difficult to achieve; PA1 g) some DNA sequences are more difficult to obtain than others; and PA1 h) resolution of multiple DNA fragments of different size is necessary to detect the presence or absence of a specific sequence at a candidate locus. PA1 a) it would be a cumbersome and difficult method for rapid detection of an uracil residue at a specific candidate locus: PA1 b) full size sequencing gels would be required for determination of an uracil residue at a specific candidate locus; PA1 c) sequencing gels can often be difficult to read/interpret; and PA1 d) resolution of multiple DNA fragments of different size would be necessary to detect an uracil residue at a specific candidate locus. PA1 a) the method is dependent on the formation of heteroduplex molecules where a mismatched based pair is formed at the site in question. Thus, to detect a mutation in a homozygous sample, an external probe must be provided and hybridization carried out to generate the mismatch; PA1 b) the method permits detection of the position of the mismatch but not necessarily the sequence at the mismatch; and PA1 c) not all mismatches are recognised with equal efficiency. PA1 a) three primers are required to determine whether wild type or mutant sequence is present at the candidate locus; PA1 b) the method does not allow one to deduce what specific sequence is present at a candidate locus; and PA1 c) the annealing conditions for the ARMS method have to precise, thus the method is difficult to transfer in many cases and has to be optimised for each mutation investigated. PA1 i) introducing a modified base which is a substrate for a DNA glycosylase into said candidate locus at one or more preselected positions; PA1 ii) excising the modified base by means of said DNA glycosylase so as to generate an abasic site; PA1 iii) cleaving phosphate linkages at abasic sites generated in step ii); and PA1 iv) analysing the cleavage products of step iii) so as to identify in said target nucleic acid sequence the presence or absence of said particular nucleic acid sequence at said candidate locus.
2) DNA sequencing
DNA sequencing allows detection of a particular sequence at any candidate locus. The main methods of DNA sequencing are the Sanger method (Sanger, F. and Coulson, A. R. (1975) J. Mol. Biol. 94, 444-448), also known as the dideoxy method or chain termination method, and the Maxam Gilbert method (Maxam, A. M. and Gilbert, W. (1977) Proc. Natl. Acad. Sci. USA 74, 560-564), also known as the chemical method.
While DNA sequencing is the ultimate way of determining if a particular sequence is presence or absence at a particular candidate locus, it suffers from a number of drawbacks as follows:
3) Uracil interference
A method has been described whereby uracil is incorporated into an amplified DNA molecule randomly and at a low level. This is achieved by amplifying the DNA in the presence of the normal DNA precursor nucleotides and dUTP. The ratio of dTTP to dUTP is chosen so that in the amplification process dUTP is occasionally incorporated opposite an adenine residue on the template strand while dTTP is incorporated opposite adenine residues at a much higher frequency. This results in a population of products bearing a low level of uracil residues randomly distributed throughout the amplified molecules. Treatment of the amplified products with uracil glycosylase and cleavage of the abasic site results in cleavage of the molecules at the position of incorporation of the uracil residues. Because the uracil was incorporated randomly opposite adenine residues at a low level, different molecules will be cleaved at different points depending on where the uracil residues were incorporated. Thus labelling of one of the primers used in the amplification process and separation of all of the cleavage products on a DNA sequencing gel produces a ladder of fragments that allows the determination of the total number of positions of uracil incorporation in one strand of the amplified DNA sample (Tu, W. T. and Struhl, K. Nuc. Acids Res., (1992) 20, 771-775. Devchand P. R. et al., Nuc. Acids Res. (1993) 21, 3437-3443).
The main application of the approach outlined has been for DNA footprinting (a method used to identify the bases in DNA to which particular proteins bind).
The uracil incorporation method can be used to determine the location of the total number of uracil residues in an amplified DNA sample. However,
4) Use of mismatched nucleotide glycosylases
Two accounts of the use of mismatched nucleotide glycosylases have been reported for detection of point mutations (Lu, A-L and Hsu, I-C, Genomics (1992) 14, 249-255 and Hsu, I-C., et al, Carcinogenesis (1994)14, 1657-1662). The glycosylases in question are the E. coli Mut Y gene product which releases the mispaired adenines of A/G mismatches efficiently and A/C mismatches inefficiently by regular glycosylase action and the human thymidine DNA glycosylase which cleaves at Gfr mismatches. These enzymes have been used for mutation detection in amplified heteroduplex DNA molecules where mismatches are present. Labelling of one of the primers used in the amplification reaction permits detection of the position of the mismatch after glycosylase treatment, cleavage of the abasic site and resolution of the fragments by gel electrophoresis.
There are several problems with this method as follows:
5) Other methods based on cleavage of mismatched base pairs
Several other methods based on the cleavage of mismatched base pairs have been described for detecting point, deletion and insertion type mutations. These include chemical cleavage at mismatched base pairs, heteroduplex detection based on the slower migration thereof during gel electrophoresis relative to homoduplex molecules which migrate faster. RNAse cleavage of mismatches in RNA:DNA hybrids and cleavage of mismatches by enzymatic means.
All of the above methods can detect mutations in heteroduplex molecules and allow the approximate position of the mutation in the nucleic acid molecule to be determined (except in the case of the heteroduplex electrophoresis retardation method which only informs of the presence of a mutation in the sample). However, these methods only work on heteroduplex DNA and do not allow one to deduce what specific sequence is present at a candidate locus.
6) Ligase chain reaction
The ligase chain reaction (LCR) is a probe amplification method that can be used for detecting the presence or absence of a particular target sequence at a candidate locus. It utilises the enzyme DNA ligase to join two pairs of oligonucleotides that hybridise adjacent to one another on the denatured target DNA strands. The enzyme forms a phosphodiester link between the two oligonucleotides, provided that the oligonucleotides at the junction correctly hybridised with the template. Thus an exact match between the oligonucleotides and the target sequence at the junction permits ligation of the oligonucleotides resulting in the formation of a larger product which is the cumulative size of both the oligonucleotides. Multiple cycles of annealing, ligation and denaturation results in the exponential amplification of the larger product. Thus detection of the larger product indicates the presence of a sequence at the candidate locus while absence of the larger product means that there were differences between the oligonucleotide sequences and the sequence of the template DNA.
While this method has good potential for detecting particular sequences at candidate loci and also offers high throughput of samples, it does not allow one to deduce what specific sequence is present at a candidate locus. In addition, the method requires considerable effort to optimise the process.
7) ARMS method
By designing appropriate primers for use in the polymerase chain reaction, it is possible to detect the presence or absence of a specific sequence at a candidate locus. In this method known as the amplification refractory method (ARMS), primers are designed so that amplification of a target sequence only occurs if there is a perfect match between the 3' end of the primers and the target sequence. Thus if a pair of primers are designed so that one primer is complementary to a given sequence of the target sample while the partner primer is designed so that its 3' end is complementary to the wild type sequence at the candidate locus, then this pair of primers will only produce a product on amplification if the wild type sequence is present at the candidate locus. If a third partner primer is designed so that its 3' end is complementary to the mutant sequence at the candidate locus, then this primer if used with the primer complementary to the given sequence of the target sample will only produce a product on amplification if a mutant sequence is present at the candidate locus. This method suffers from a number of problems as follows:
Thus, it will be appreciated that there is a need in the nucleic acid diagnostics field for a robust method for detection of specific sequences at candidate loci that allows rapid and high throughput of samples.
Methods in Molecular Biology, Vol. 9, 1991, p51-68 describes various rapid methods for detecting polymorphic markers in DNA. The polymorphisms are detected by amplification of the DNA surrounding and including the locus of interest and mismatch detection at that locus. One of the methods involves cleaving the DNA phosphate linkages at sites of mismatches following modification and subsequently analysing the cleavage products so as to identify the presence or absence of a particular polymorphism in the genomic DNA. The alternative methods described therein rely on the differential hybridisation and/or extension of oligonucleotide primers to sites of polymorphisms dependent on whether a mismatched base pair is generated during the hybridisation step. These methods rely on analysing the hybridised or amplified products so as to identify the presence or absence of a particular polymorphism in the genomic DNA. The methods described do not involve the introduction of a modified base, which is a substrate for a DNA glycosylase, into the amplified DNA and do not involve the excision of such a modified base by the DNA glycosylase.